Mitochondria are multivariate signal processors of cytoplasmic [Ca2+] flux. Mitochondrial [Ca2+] participates in cellular energy production via activation of mitochondrial enzymatic complexes yet can also promote different modes of cell death. Mitochondria exquisitely maintain cytosolic Ca2+ gradient via numerous pathways. Dysregulation of mitochondrial Ca2+ uptake has been linked to numerous cellular dysfunctions including chronic oxidative burden, autophagy and sensitization for cell death. High cytoplasmic [Ca2+] facilitates mitochondrial Ca2+ accumulation via an unknown until 2011 uniporter. Recently, we demonstrated that mitochondrial Ca2+ uptake 1 (MICU1) and mitochondrial Ca2+ uniporter regulator 1 (MCUR1) negatively and positively control the mitochondrial Ca2+ uniporter pore submit (MCU) activity under resting and active state. MCU interacts with MICU1 and MCUR1 separately and forms different MCU complexes at the mitochondrial inner membrane. Given the critical importance of mitochondrial Ca2+ for the MCU interactome, we hypothesize that MCU is a major hub of the MCU complex and interacts with a network of proteins to form the MCU complex. Further, MCU interacting components are crucial for MCU complex assembly and will be dependent on cytosolic Ca2+ levels. The goal of the proposal is to identify and validate mammalian MCU binding partners by the application of state-of-the-art proteomic technologies and super-resolution imaging. To accomplish the identification of the functional mitochondrial Ca2+ flux interactome, four highly qualified investigators are recruited. Muniswamy and Merali laboratories will identify the MCU interacting partners and focus on the identity and function of molecular interactions with MCU. Further, the physical interaction of the MCU interactome will be tested in a reconstituted system (Mancia lab). Finally, the Caplan lab will visualize the MCU interactome complex with super-resolution imaging. The critical next step in mitochondrial biology requires the collaboration of these four investigators and technologies thus providing an ideal strategy for future collaborations of macromolecular interactions. The results will greatly enhance our understanding of the MCU complex and its role in mitochondrial Ca2+ influx during the normal and active states of the cell.